UC Riverside

Telomeres are DNA-protein complexes that maintain the integrity of linear chromosomes. Telomeres protect the chromosome ends from being recognized as a DNA double strand break by the DNA damage checkpoint, and prevent chromosome rearrangements. Hence, telomeres are crucial to defending against genomic instability, a hallmark of cancer cells. The work presented in this dissertation will focus on the regulation of activities involving the essential telomeric DNA binding protein Cdc13. In S. cerevisiae, the single-strand telomere binding protein Cdc13 is important for protecting chromosome ends. Previous studies indicate a role for Cdc13 in both telomere end protection and recruitment of telomerase to the telomere. The role of CDC13 in telomere end protection has been elucidated using the temperature sensitive allele, cdc13-1. In cdc13-1 mutants the C-rich telomere strand is lost when cells are grown at high temperatures. The accumulation of single-stranded DNA at the telomere leads to a DNA damage checkpoint response and loss of cell viability. We describe a screen conducted to identify activities involved in telomere C-strand loss, in which we identified two novel alleles of RAD24. Rad24 is an alternate Rfc1 subunit, and functions to load the 9-1-1 checkpoint clamp onto sites of DNA damage. In each rad24 allele, the transposon used in the screen is inserted within the RAD24 coding region and results in production of two amino terminal truncations of Rad24. Here, we show that an intact Rad24 amino-terminus is necessary for its checkpoint function. The rad24-2 allele increases the frequency of obtaining cdc13-1 cells capable of growth at high temperatures. This rad24-2 allele combined with acquired telomere amplification facilitates growth of cdc13-1 cells at high temperatures. The next work presented is an analysis of a Cdk1-mediated phosphorylation of Cdc13 on residue T308. Strains with a T308A mutation at that phosphorylation site had short telomeres, suggesting a role for the Cdk1- mediated phosphorylation of Cdc13 at T308 in telomerase recruitment. In the final work, we describe an analysis of the interactions between Cdc13 and the DNA polymerase α complex. The association of Cdc13 with Pol1, the catalytic subunit of the DNA polymerase α complex is proposed to recruit the conventional DNA replication to participate in C-strand synthesis of the new G-rich strand extended by telomerase. We demonstrate that the Pol1 and Cdc13 as well as Pol12 and Stn1 interact directly, and loss of these interactions using the pol1-236 and pol12-40 alleles results in elongated telomeres. Thus, the data shown here will provide insight on how Cdc13 functions both to protect chromosome ends and to facilitate telomere replication.